Overview

Annexin V-FITC Apoptosis Detection Reagent ab14082 can be used in a 10 min, one-step staining procedure to detect apoptosis by staining phosphatidylserine molecules which have translocated to the outside of the cell membrane. Analysis is by flow cytometry or fluorescence microscopy.

This Annexin V-FITC reagent can be used with Annexin V binding buffer ab14084 (see protocol section for instructions). The Annexin V-FITC reagent is also available as part of Annexin V-FITC apoptosis kit ab14085.

This protein is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade.

Involvement in disease

Pregnancy loss, recurrent, 3

Sequence similarities

Belongs to the annexin family.Contains 4 annexin repeats.

Domain

The [IL]-x-C-x-x-[DE] motif is a proposed target motif for cysteine S-nitrosylation mediated by the iNOS-S100A8/A9 transnitrosylase complex.A pair of annexin repeats may form one binding site for calcium and phospholipid.

I can confirm that ab14082 (5X Annexin V-FITC Apoptosis Detection Reagent) and ab14084 (10X Annexin V Binding Buffer) are the same reagents as in the kit ab14085(Annexin V-FITC Apoptosis Detection Kit).

Please note that ab14082 (5X Annexin V-FITC Apoptosis Detection Reagent) and ab14084 (10X Annexin V Binding Buffer) are more concentrated and need need to be diluted to 1x before using them.

I hope this information is helpful and wish you and your customer a good week.

Both Annexin V-EGFP Apoptosis Detection Reagents (ab14152 and ab14082) contain a protocol booklet in their datasheets. These are the recommended protocols to use along with the Propidium Iodide reagent.

The concentration of this product (ab14082) is 0.15 mg/ml, which is equivalent to 0.15 µg/ul. This is added to 500 µl makes it 0.15 µg/500 µl, which is 0.3 µg/ml. The PI is 1 mg/ml which is equivalent to 1 µg/µl, and in 500 µl it would make 2 µg/ml. (But note that the PI reagent actually has to be diluted to 250 µg/ml before use, which would mean that if this is done, the final concentration of PI in the 500 µl solution would be 0.5 µg/ml).

I hope this helps and if I can assist further, please do not hesitate to contact me.

Thank you for contacting us for technical support, I'm sorry to hear you are experiencing problems with ab14082, I am trying to find out from the lab the function of the binding buffer and as soon as I find out I will forward to you this information.
Here is the recommended protocol with this antibody, I will ask my colleagues to add it to the datasheet:
A. Incubation of cells with Annexin V-FITC:
1. Induce apoptosis by desired methods.
2. Collect 1 x 105 cells by centrifugation.
3. Resuspend cells in 500 µl of 1X Annexin V Binding Buffer.
4. Add 1 µl of Annexin V-FITC and 1 µl Propidium Iodide.
5. Incubate at room temperature for 5 min in the dark.
Proceed to B or C below depending on method of analysis.
B. Quantification by Flow Cytometry:
Analyze cells by flow cytometry (Ex. = 488 nm; Em. = 530 nm) using FL1 channel for detecting Annexin V-FITC staining and FL2 channel for detecting PI staining.
For adherent cells, trypsinize and gently wash cells with serum-containing medium before incubation with Annexin V-FITC.
C. Detection by Fluorescence Microscopy:
1. Place the cell suspension on a glass slide, and cover with a glass coverslip.
For analyzing adherent cells, grow cells directly on a coverslip. Following incubation, invert coverslip on a glass slide and visualize cells. The cells can also be washed with 1X Annexin V Binding Buffer and fixed in 2%
formaldehyde before visualization. (Cells must be incubated with Annexin V-FITC before fixation because any cell membrane disruption can cause nonspecific binding of annexin V to PS on the inner surface of the cell
membrane.)
2. Observe the cells under a fluorescence microscope using a dual filter set for FITC and rhodamine, or separate filters.
Cells that have bound Annexin V-FITC will show green staining on the plasma membrane. Cells that have lost membrane integrity will show red PI staining throughout the nuclei and a halo of green staining (FITC) on the
plasma membrane.
I hope this protocol will help you and will forward to you the information about the binding buffer as so as I receive it, Thank you for your patience,

Thank you for your enquiry. The associated concentration for the Annexin V- FITC conjugate in the Annexin V- FITC Apoptosis Detection Reagent Kit is 250 ug/ml. Please contact us again if you require further assistance.